Prof. Dr Beate M. Henz, Department of Dermatology, Charité, Campus Virchow, Humboldt University, Augustenburgerplatz 1, 13344 Berlin, Germany Tel.: 49 30 450 65001 Fax: 49 30 450 65900 e-mail: firstname.lastname@example.org
Abstract: Until recently, mast cells have been viewed primarily as harmful because of their key role as effector cells of allergic and potentially lethal anaphylactic reactions. Their contribution to human health appeared instead to be limited to the elimination of parasites. There is, however, growing evidence for additional beneficial functions of mast cells, particularly regarding the initiation of acquired immune reactions. Thus, mast cells can phagocytize diverse particles, take up antigens, and express a number of receptors, particularly MHC class I and II antigens, ICAM-1 and -3, CD43, CD80, CD86 and CD40L which allow them to interact with T and B lymphocytes. They can also secrete numerous cytokines that induce and enhance recruitment and functions of lymphocytes. Finally, there is good evidence that mast cells present e.g. pollen and bacterial antigens, respond to bacterial superantigens, but fail to react to endogenously produced antigens or superantigens. Mast cells can also activate B cells directly to produce IgE, but this activity and the ability to produce IL-4 or IL-13 is restricted primarily to basophil leukocytes and mucosal mast cells. Finally, recent evidence attributes a pivotal role to the cells in natural immunity to bacteria. There is also emerging evidence that mast cells can downmodulate the immune response. While these data require further clarification, the basic ability of mast cells to initiate innate and acquired immune reactions can no longer be questioned.
Mast cells (MC) are ubiquitously distributed, resident connective tissue cells. They are particularly frequent in close proximity to epithelial surfaces in the skin, the respiratory system and the gastrointestinal mucosa where they are strategically located for optimal interaction with the environment and for their putative functions in host defense (1, 2). For many decades, MC have been viewed primarily as effector cells of anaphylactic reactions and thus as potentially harmful (3). Recently, MC have also been to shown to exert beneficial functions such as in tissue repair (4) and in acquired and innate immune responses against foreign molecules and infectious agents (5, 6). Data published so far on the role of MC in the initiation of immune recognition are in part incongruous or contradictory. This can be viewed as partly due to the models employed and the great functional variability of MC with regard to species, location within the organism and in vitro culture conditions used for their generation. MC also vary widely in their receptor repertoire and their responsiveness to diverse stimuli and pharmacological agents (1, 2).
The present article will review evidence for the ability of MC to initiate acquired and innate immune responses, scrutinizing in particular whether MC fulfill all basic prerequisites for antigen presentation to T-cells (Table 1). Special emphasis will be given to the human system and to skin MC, as far as data are available, and concrete examples will be presented for immune responses in which MC have been shown to play a specific role.
Table 1. Basic functional prerequisites for MC to be classified as APC in acquired immunity
Phagocytosis and antigen uptake
MC arise from CD34+ myeloid stem cells in the bone marrow, circulate in the blood as not further characterized precursors among adherent monocytic cells, and home into the tissue where they differentiate under the influence of specific growth factors (1, 7). These constitute primarily stem cell factor (SCF) and nerve growth factor in humans and, in addition, IL-3 in the murine species (8, 9). The close ontogenetic relationship of MC to myeloid phagocytes is evident from their ability to phagocytize diverse micromolecular and particulate materials like colloidal gold, ferritin, aggregated IgG, yeast cells, bacteria, red blood cells and latex beads (reviewed in 10), but this ability decreases with increased maturity of the cells. Thus, we observed that during in vitro rat peritoneal MC development, the number of cells phagocytizing latex beads dropped from 98% to 77% during 2 weeks of culture, and only 7% of mature peritoneal MC had taken up latex beads under the same conditions (11). The massive uptake of latex particles by immature (4-day culture) rat MC within 30 min of incubation is illustrated in Fig. 1. The sausage-link nature of interconnections between electrondense MC granules becomes evident due to the fact that they are loaded with the particles. In immature human MC cultured for 3 weeks from their peripheral precursors, only 50% were able to take up latex beads (12).
During phagocytosis, induction of mediator release may occur in a dose-dependent fashion as early as during the first 15 min of incubation, as shown for histamine, β-glucuronidase and eosinophil chemotactic factor (leukotriene (LT) B4) secretion from rat peritoneal MC in response to latex, a process that occurs more efficiently on exposure to zymosan, and even more so to zymosan coated with complement (10). Apparently, only IgG receptors IIb2 and III are involved in phagocytosis of antigen-coated erythrocytes (reviewed in 5). Very recently, it was also shown that murine MC uptake of IgE complexed to a pollen allergen (Lol p1) was enhanced compared to similarly complexed IgG (13).
A direct contact of MC with lymphocytes is a fundamental requirement for antigen presentation. Such heterotypic contacts have indeed been demonstrated by electron microscopy for human nasal mucosal MC (14).
MC provide all basic prerequisites for interacting with lymphocytes: A number of the preformed or newly generated MC-mediators, including histamine, leukotrienes, platelet activating factor (PAF), TNF-α, IL-1, IL-4, IL-6, IL-8, IL-13 and VEGF, induce the expression of endothelial adhesion molecules essential for lymphocyte homing (reviewed in 2 and 15) (Fig. 2). This is evidenced best in urticaria, the prototype of mast cell dependent disease: Sequential biopsies of elicited wheals exhibited a rapid upregulation of P- and E-selectin (which mediate leukocyte rolling), followed by upregulation of ICAM-1 and VCAM-1 which induce firmer adhesion of cells (reviewed in 16). MHC class II and TNF-α are also upregulated in endothelial cells in urticaria (17, 18).
Human MC have recently been shown to generate a number of specific chemotactic factors required for immigration of lymphocytes into tissue. Thus, human leukemic (HMC-1) and skin MC can generate, store and secrete the CXC chemokine IL-8 (19–21), a potent chemotactic factor for lymphocytes (22) (Fig. 2). Lymphotactin, a C-chemokine whose functions are highly restricted to CD8+ T cells, is also produced by HMC-1 and by murine MC (23), and human lung and cultured bone marrow-derived MC (BMMC) secrete IL-16, a chemoattractant for CD4+ lymphocytes (24). MIP-1β, another chemokine produced predominantly by MC, has furthermore been shown to play a pivotal role in T-lymphocyte recruitment from lymph nodes to the skin during sensitization (25, 26).
MC can not only attract lymphocytes, but they can conversely migrate towards these cells, as evidenced from data showing their migration to lymph nodes in the context of contact sensitivity reactions (25, 26). For this migration, they are equipped with integrins (Fig. 3), which enable them to interact with endothelial cells and with connective tissue components like fibronectin, laminin, collagens I, II and IV, and vitronectin (reviewed in 27, 28). In recent years, a number of chemotactic factors for human MC have been identified (reviewed in 4), but their specific production by lymphocytes has not yet been investigated. IL-8 would be a possible candidate among these factors since human HMC-1 and skin MC have been shown to express the CXCRI and II receptors for this chemokine (29).
Finally, IL-4-primed BMMC can induce resting spleen cells to produce activated, IL-12 and IFN-γ producing B- and T-lymphocytes (30), underlining the functional relevance of MC-lymphocyte interaction.
Major histocompatibility antigen expression on mast cell
After antigen uptake and processing, a major prerequisite for MC antigen presentation to T-cells is an interaction of these cells via the major histocompatibility (MHC) complex and the T cell receptor (TCR) on CD4+ or CD8+ lymphocytes (Fig. 3).
Expression of MHC class I molecules on all MC subsets, including cultured murine BMMC and human MC in different organs (lung, liver, uterus, skin), has clearly been demonstrated in the past (31, 32). Furthermore, bacterial antigens can be presented by MC to T cells via MHC-I (33) (see also below).
The situation is far more complex with regard to MHC class II expression. The highly divergent data available so far are summarized in Table 2. Thus, most resting murine and human MC fail to express MHC class II molecules. They are, however, upregulated in MC isolated from infected tissue or during exposure to specific stimuli. Thus, T-cell supernatants, IFN-γ and LPS have been shown to transiently increase MHC class II expression in up to 90% of murine MC progenitors, rat peritoneal MC and BMMC (32, 34, 35). On stimulation of HMC-1 cells with IFN-γ, an increase of MHC II expression from 21% at baseline to 81% was noted after 3 days (36). These latter authors found no effect with IL-4, while we were able to show a time and dose-dependent upregulation of HLA-DR, -DP and -DQ molecules from 0% at baseline up to a 22% maximum of positive cells by IFN-γ, TNF-α, or IL-4 (IFN-γ=TNF-α >IL-4). Downregulation can be observed on extended incubation within 3 days of culture with the more potent stimuli (37). Eighty percent of CD34, c-kit and CD13 positive human progenitors have also been found to express HLA-DR, but lost this expression during 3 weeks of differentiation in cultures with SCF and IL-6 (38). Interestingly, in skin sections from various types of inflammatory and immunological diseases including atopic dermatitis, we were unable to detect any MHC class II positive MC (31), underlining that expression of these molecules depends on the state of MC-maturation and -activation.
Table 2. Percentage of MC expressing MHC class II antigens (for literature, see text)
MC as APCs
There is also increasing evidence that MC can present antigen to T cells (Fig. 3) although their capacity to do so seems restricted. Initial studies were performed with murine MC. Initially, rat peritoneal MC were shown to present antigen to a PPD specific T-cell line (39). Further data were generated from murine BMMC whose differentiation was induced during culture with IL-3. These cells lack Ia antigens and antigen presenting capacity, but on addition of IL-4 and IFN-γ, these properties were upregulated, and GM-CSF in combination with IL-4 further enhanced, whereas IFN-γ completely abrogated, the antigen-presenting capacity of these cells (40). Expression of MHC class II was also shown in this model to be the restricting element for the presentation of ovalbumin (32). Furthermore, these murine BMMC were shown to present exogenous antigens and superantigens, but not endogenously produced peptides and self-superantigen, as holds for other nonprofessional antigen-presenting cells (41). In more recent studies, it was demonstrated that BMMC can present the major allergen of grass pollen Lolium perenne (Lol p 1) to a specifically sensitized T-cell hybridoma. Complexing of the antigen with IgE greatly enhanced the efficiency of this process, compared to antigen-IgG complexes; an irrelevant antigen (dinitrophenyl) was ineffective (13). Finally, there is recent evidence that murine MC can stimulate IL-2 production in a CD8+ T-cell hybridoma specific for a bacterial cytoplasmatic protein (Crl) (42).
Only few data are available indicating that human MC have similar capacities. Thus, HMC-1 cells as well as human CBMC have been shown to internalize a number of clinically relevant bacteria, with subsequent activation of protein kinase C (PKC) and secretion of TNF-α (43, 44). The same cells were able to stimulate IL-2 production by CD4+ T-cell hybridoma via the staphylococcal enterotoxin B (SEB) and TSST1 superantigens (44).
A number of costimulatory molecules have been identified in recent years. They play an important role in regulation of the immune response and enhance the efficiency of antigen presentation via the MHC-TCR complex by inducing lymphocyte proliferation (Fig. 3). They also play a decisive role with regard to the type of immune response that will develop following antigen presentation. The costimulatory molecules identified so far on MC are shown in Fig. 4.
MC have been reported to express ICAM-1, ICAM-3, β2-integrins, and members of the B7 family (CD80, CD86), enabling them to interact with endothelial cells, lymphocytes and fibroblasts.
Expression of ICAM-1 (CD54) antigen and mRNA by human MC was already described in 1991 (45) and was subsequently demonstrated on rat peritoneal MC and murine BMMC as well as on human skin, lung, cardiac, conjunctival and HMC-1 MC (36, 39, 46–50). ICAM-1 expression is upregulated by phorbol myristate acetate (PMA), IL-4, IL-13, IFN-γ, TNF-α and retinoic acid (RA), and its downmodulation is induced by dexamethasone (28, 36, 45, 51–53) (Table 3).
Table 3. Summary of data on expression, modulation and induction of cytokine production of selected adhesion molecules on skin MC and HMC-1 cells (for further details and references, see text)
Expression of ICAM-3, another member of the immunoglobulin superfamily of adhesion molecules with high homology to ICAM-1 and -2, differs from ICAM-1 in that it is restricted to cells of the immune system. Thus, ICAM-3 is highly expressed on Langerhans and dendritic cells, is found on resting lymphocytes, neutrophils and monocytes, is differentially expressed during lymphoid differentiation and/or activation, but is not found on resting endothelial and epithelial cells (55–58). While one group detected this molecule on human lung, but not on cutaneous MC (foreskin and mammary skin) (48), we have found that >95% of HMC-1 cells and a variable percentage (8.6–71.4%) of isolated and highly purified human skin MC express ICAM-3 (Table 3) (58). ICAM-3 is involved in the induction of homotypic aggregation and the expression and secretion of IL-6 and IL-8 (58). It is modulated by RA and vitamin D (up) and PMA (down) (59 and data submitted).
LFA-1 (CD11a/CD18) belongs to the β2-integrins, is restricted to leukocytes, and serves as ligand for ICAM-1 and -3. It has been described on rat peritoneal MC (39), is expressed at low levels in HMC-1 and skin MC, at much higher levels in the more mature HMC-1 clone 5C6 and in cord-blood derived MC (49), is upregulated in the presence of LTB4, and PMA, and is downregulated by RA and dexamethasone (Table 3) (53, 60).
CD80 and CD86 interact with CD28 on lymphocytes and cytotoxic T-cells and are upregulated by GM-CSF. They have been studied on MC only in the context of antigen presentation (41, 44).
The potential relevance of leukosialin (CD43) within the immune system has as of yet not been clarified in detail, but the molecule is able to induce lymphocyte proliferation and is expressed also on monocytes, neutrophils and other hematopoietic cells where it is known to transduce stimulatory signals (reviewed in 61). CD43 has been detected on human lung, skin and HMC-1 MC, is involved in a signaling pathway leading to cellular aggregation and cytokine production of HMC-1 cells (47, 48), and is upregulated in inflammatory diseases like psoriasis, urticaria and lichen planus (unpublished). It can induce MC homotypic aggregation (47), is upregulated by RA (54), and is downmodulated by PMA (62, 63) (Table 3).
CD40 ligand (CD40L) is a glycoprotein that is expressed on the surface of activated helper T cells, basophils, and eosinophils. Binding of CD40L to its receptor CD40 on the B-cell surface induces B-cell proliferation and adhesion (64). In the presence of interleukin-4, CD40L induces B-cell proliferation and a switch to the ε-germline, resulting in IgE production (65). The potential significance of this molecule on MC became apparent when HMC-1 cells and purified human lung MC were shown to express CD40L (66). Furthermore, HMC-1 cells stimulated B-cell IgE production on addition of IL-4 (66). These data have recently been confirmed in patients with perennial allergic rhinitis whose nasal MC, in contrast to those of patients with chronic infectious rhinitis, expressed higher levels of FcεRI, CD40L, IL-4, and IL-13 and were exclusively able to induce IgE synthesis by purified B cells in the presence of Der fII (mite antigen) (67). We were unable to demonstrate CD40L expression on unstimulated HMC-1 cells (unpublished) and in skin biopsies from normal donors and patients with urticaria, atopic dermatitis and scabies, although an increase in CD40L was found in the inflammatory infiltrate in the latter two conditions (68). The apparent variability of CD40L expression in MC is underlined by data showing that human basophils, but not cord blood-derived MC, expressed detectable CD40L and released soluble CD23 (69). Studies revealing the molecular mechanisms of CD40L regulation in MC may shed light on the reasons for these conflicting data.
Human MC have been reported to secrete a broad spectrum of cytokines which may contribute to immunological reactions by affecting lymphocyte growth, recruitment and function, including IL-1β, -3, -4, -5, -6, -8, -10, -12 (only the p35 chain), -13, -16, TNF-α, GM-CSF, TGF-β, IFN-γ and VEGF (reviewed in 3, 4) (Fig. 2). IL-2 protein has never been demonstrated in human MC, underlining the fact that in most circumstances, MC express a pattern of cytokines corresponding to that of TH2 lymphocytes.
Cytokine production by MC is highly dependent on the type of MC studied. Thus, immature HMC-1 cells fail to secrete IL-3 and -13, upregulate IL-4 mRNA only transiently, and induce secretion of only small amounts of IL-4 on exposure to stimuli that induce a wide range of other cytokines (70 and unpublished). Normal cutaneous MC fail to secrete IL-3, -4, -5, and -13 on stimulation with anti-IgE, PMA plus the Ca-ionophore A23187, substance P or compound 48/80 (21 and unpublished). In agreement with this, cord blood-derived MC fail to secrete significant amounts of immunoreactive IL-4 and IL-13 on IgE-dependent stimulation (69). In contrast, MC located in the lung and nasal mucosa have been shown to produce IL-4, albeit in small amounts in isolated lung MC (71). Diverse allergens (bee venom phospholipase, Der p I and II, Schistosomal protease) have all been shown to induce IL-4 secretion also in nonsensitized lung MC (72, 73). Earlier work also claimed expression of IL-4 in skin MC (reviewed in 3), but this work has not been confirmed subsequently (see above).
The obvious variability in the ability of MC to produce IL-4 may be due to disease-dependent microenvironmental changes. This concept is supported by cultures of murine BMMC since cells kept in IL-3 produce IL-4 whereas IL-12 is secreted by those kept in SCF (74). These data underline the highly restricted and tightly regulated production of IL-4 in MC, as also demonstrated in studies comparing the activation-response elements of mast cell and T-cell nuclear extracts which differ at least in part (75, 76).
In several experiments using cocultures of MC and activated T lymphocytes or their membranes, MC have been shown to produce cytokines. Thus, HMC-1 cells release histamine, TNF-α and IL-8 in this setting (77–79), and increased IL-4 mRNA transcription has been observed in murine BMMC through an MHC-II dependent pathway (79). Murine BMMC have also been shown to stimulate B-cell blast formation, proliferation and IgM production on coincubation (80). This response is due to soluble factors that have so far not been identified; IL-4, IL-6, chondroitin and heparin could be excluded (80).
The importance of cytokines in determining MC function is highlighted by recent findings in murine MC. Thus, IL-12 induces rat peritoneal MC to produce IFN-γ while this fails to occur on stimulation with anti-IgE or LPS (81). Furthermore, IFN-γ production has recently been demonstrated in MC from psoriatic skin and in PMA-stimulated HMC-1 cells (82). These findings are of particular interest since they suggest that MC can induce TH1 in addition to TH2 responses of the immune system, depending on the type of stimulus to which they are exposed.
Role of MC in innate immunity
Recent in vivo studies employing genetically MC-deficient mice showed that MC are essential for mounting efficient innate immune responses against bacterial infections. Thus, Malaviya et al. (33, 83) reported that MC-deficient KitW/KitW-V mice are less efficient in clearing and surviving experimentally induced enterobacterial infections as compared to wild-type mice or MC-reconstituted KitW/KitW-V mice. Impaired killing of bacteria in MC-deficient mice was directly correlated with fewer neutrophils at the sites of infection, most likely as a result of lower levels of the MC-derived PMN chemotactic activity induced by TNF-α in these mice (33, 83). Along this same line, Echternacher et al. (84) found that MC-deficient mice exhibit dramatically increased morbidity and mortality after cecal ligation and puncture (CLP, a model for acute septic peritonitis), compared to normal control mice. Adoptive transfer of MC to the peritoneum substantially protected MC-deficient mice from the lethal effects of CLP (85).
The mechanisms involved in MC activation in the context of innate immune responses is slowly evolving (86, 87) (Table 4).
Table 4. Pathological conditions and examples of diseases in which MC exert immune functions via different mechanisms
As discussed earlier, MC can not only phagocytize bacteria, but they recognize infectious agents through specific receptors which are present on their surface (88). FimH, a mannose-binding lectin, expressed by many enterobacteria including E. coli, K. pneumoniae and S. typhimurium, binds to and mediates MC secretion of TNF-α via the surface receptor CD48 (89) (Fig. 4). CD48 which is localized to plasmalemmal caveolae in MC, appears to facilitate the formation of bacteria-encapsulating caveolar chambers shown to be involved in bacterial entry into MC (90).
In the CLP model, mice lacking the complement components C3 showed a markedly increased mortality after CLP, compared to wild-type mice (85). MC-activation was furthermore significantly reduced and associated with diminished intraperitoneal levels of TNF-α, fewer neutrophils and increased bacterial load in C3−/− mice (85). Prior intraperitoneal injection of C3 into C3−/− mice normalized all of these defects and restored protection from mortality after CLP (85). More recently, C3b receptor (CD35)-deficient mice (Cr2−/−) were shown to exhibit impaired MC-activation and neutrophil recruitment, associated with reduced bacterial clearance and survival after CLP (85). Peritoneal mouse MC also express the complement receptor 3 (CR3, Mac-l, CD11b/CD18), and CR3-deficient mice exhibit decreased levels of the MC-mediator histamine as well as reduced neutrophil recruitment and survival after CLP (91).
TNF-α which activates endothelial cells and induces leukocyte recruitment in vivo, has been demonstrated to provide antibacterial protection in MC-deficient mice, as does reconstitution with MC (84). Furthermore, TNF-α deficient mice exhibit increased mortality in acute septic peritonitis, compared to-wild-type mice (92). MC which store TNF-α in secretory granules, have been shown to release this cytokine after incubation with bacteria, and only wild-type and MC-reconstituted KitW/KitW-V mice, but less so MC-deficient KitW/KitW-V mice, exhibit local TNF-α release after bacterial challenge (33, 83). In addition, MC also release significant amounts of the potent chemotactic factor LTB4 and the smooth muscle contracting LTC4 on incubation with E. coli, and neutrophil influx and bacterial clearance after intraperitoneal injection of E. coli were significantly decreased in mice pretreated with a leukotriene synthesis inhibitor (93). Taken together, the main function of MC in innate immune responses appears to be initiation of inflammation and recruitment of neutrophils, with resulting bacterial killing, via leukotrienes and TNF-α.
Further support for the concept that MC are crucially involved in innate immunity and that these findings may have therapeutic implications, was recently provided with in vivo studies. Thus, repetitive injections of C57BL/6 mice with the MC growth factor stem cell factor (SCF) not only increased the number of MC, but also significantly improved survival of these mice after induction of acute septic peritonitis by CLP (92). SCF only improved MC-dependent innate immunity in the presence of MC (wild-type mice and MC-reconstituted KitW/KitW-V mice), but not in MC-deficient KitW/KitW-V mice, demonstrating that the effect of SCF treatment reflected, at least in part, the modulation of MC numbers and/or function by SCF (92).
MC immune function in specific diseases
Apart from the data on innate immunity, the well-documented activity in the elimination of parasitic infestations (94) and their potential role in atopy on the basis of effects on allergen presentation and IgE synthesis (13, 66, 72, 73), evidence for the immune function of MC in other pathological conditions is weak or fragmentary (Table 4).
For delayed type hypersensitivity reactions (DTH), a series of studies in a murine model have suggested that mast cell activation and serotonin release is crucial for the induction of contact sensitivity (95). Subsequent controversial findings with mast cell deficient and reconstituted mice were in part resolved in that MC were found to be essential only in a macrophage-independent DTH elicited with albumin and incomplete Freund’s adjuvant, whereas classical DTH, as examplified by the tuberculin reaction, needs vasoactive amines only during its early phases, and these can also come from other sources such as platelets (96) (Table 5). Recent studies with mast cell and TNF-α deficient mice underline the essential role of mast cell derived TNF-α in clinical aspects and neutrophil recruitment during trinitrochlorobenzene or para-phenylindiamine induced contact sensitivity (97, 98). Also, the role of MC during intestinal DTH has been confirmed in a model with doxantrazole as sensitizer (99). In humans where MC lack serotonin, studies showing evidence for a role of MC in DTH are still missing.
Table 5. Characteristics of different DTH reactions
In acute graft versus host disease (GvHD), a critical role for initial endothelial activation by MC has also been demonstrated, together with damage and disappearance of the cells. MC derived TNF-α seems to play a critical role in the associated target cell injury (100). In an interesting recent study with T-cell deficient mice, a massive increase of MC in GvHD was shown to be of donor origin, and bone marrow derived donor cells were shown to be responsible for the pathological changes normally induced by T cells in mice (101). In humans, decreased numbers of MC have been demonstrated in skin biopsies from patients with chronic GvHD (102) which suggests an activation of the cells. A direct involvement in immune responses has however not been demonstrated.
Taken together, the present data, although as yet incomplete, point to an expanding role of MC beyond their well documented role in immediate type hypersensitivity reactions where their function is primarily harmful. This may have long-term implications for the treatment of MC-dependent diseases. Hopefully, the data summarized here will stimulate further research to clarify the role of MC in innate and acquired host defense.